Pharmaceutical preparation for treating blood coagulation disorders

ABSTRACT

There is disclosed a pharmaceutical preparation for treating blood coagulation disorders which comprises purified prothrombinase factors, in particular purified prothrombin and optionally purified factor Xa as active component.

This application is a divisional of application Ser. No. 08/821,763,filed Mar. 20, 1997 now U.S. Pat. No. 5,866,122.

The present invention relates to a pharmaceutical preparation, inparticular for the treatment of blood clotting disorders, comprisingcoagulation factors which are components of a prothrombinase or of apro-prothrombinase, respectively.

Prothrombinase is an enzyme substrate complex which forms on aphospholipid surface and enables the activation of prothrombin. Bydefinition, prothrombinase consists of factor II (prothrombin),activated factor X (factor Xa), cofactor V or Va, respectively,phospholipids and calcium ions. In vivo, these factors are present as atransient complex for the activation of prothrombin and the formation ofthrombin.

A corresponding pro-prothrombinase is defined as a complex of factorswhich are present at least partially modified or activated for theformation of a prothrombinase. Pro-prothrombinase thus is to beunderstood as a proform of a prothrombinase and as complex in which oneor more components are present in their pre-stages, as zymogens or asproforms and which is formed on the basis of affinities of thecomponents to one another.

Hemophilia A arises from an X-chromosomal recessive hereditary factorVIII deficiency and becomes manifest by severe blood coagulationdisorders. For controlling acute bleedings, coagulation-active plasmaprotein concentrates are used in most cases, primarily factor VIIIconcentrates. However, in the classical treatment of hemophiliaA-patients with factor VIII preparations, antibodies to factor VIII willform in about 20% of the cases, which antibodies lead to an inhibitionof the factor VIII administered with these preparations. One then saysthat a patient has formed a functional inhibitor to factor VIII and hasdeveloped a so-called factor VIII-inhibitor hemophilia or acquiredhemophilia.

At present, several methods are used for the therapy of hemophiliaA-patients having a factor VIII inhibitor hemophilia:

1.) Treatment with high doses of a factor VIII preparation:

With this, the antibody directed against factor VIII is neutralised invivo, and the excess of factor VIII can unfold its hemostatic cofactoractivity. By repeated administration over an extended period of time,the afflicted patient is desensitized against factor VIII andsubsequently in many cases may be subjected to the common factor VIIIconcentrate therapy. This mode of procedure requires extremely largeamounts of factor VIII, is time-consuming and may involve massiveanaphylactic side effects at the onset of treatment.

2.) Treatment of factor VIII inhibitor patients with immunoglobulinpreparations which contain antiidiotypic factor VIII antibodies:

This route of therapy at present is subject to intensive research.However, no final judgment is as yet possible on the efficacy of such atreatment.

3.) Immunoadsorption:

A further complex method for removing factor VIII inhibitors is theextracorporeal immunoadsorption either on lectins which bindimmunoglobulins (protein A, protein G), or on immobilized factor VIII,to which the antibody formed against factor VIII is bound. This methodis complicated for the patient because in this case he is bound to anapheresis machine, as with the previous methods it may not lead to acessation of an acute bleeding and, furthermore, it is expensive.

4.) APCC and derivatives

At present, the therapy of choice is the administration of activatedprothrombin complex concentrates (APCC), FEIBA®, AUTOPLEX®, which can beutilized to stop acute bleedings also in patients having high inhibitortiters (cf., e.g., DE-PS 31 27 318 (2)).

Based on the activated prothrombin complex factor concentrates, acomponent which is also contained in the former, i.e. activated factorVIIa, has been suggested as a therapeutic principle for factor VIIIinhibitor patients via the extrinsic coagulation pathway. Acorresponding preparation, i.e. recombinant factor VIIa, at present isbeing clinically tested (Hedner et al., Transfusion Medicine Reviews 7(2): 78-83 (1993)). Preclinical trials, e.g. on dogs suffering fromhemophilia A, have, however, indicated that the treatment withrecombinant factor VIIa is ineffective. Similarly, also the success ratein human application is varying. A further disadvantage of recombinantfactor VIIa consists in that on account of its very short half-life invivo, the latter must be administered frequently in high doses per dayto control severe bleedings, if possible at all. In such instances it isattempted to administer factor VIIa together with antifibrinolyticagents so as to aid its effect.

In the literature (e.g. DE 44 16 180 A1) it has also been proposed totherapeutically utilize a combination of factor Xa and phospholipids forthe treatment of hemophilia A inhibitor patients. In vivo tests onfactor VIII-deficient dogs with inhibitor showed that in a suitabledosage such a combination is able to stop an acute bleeding. Thetherapeutic spectrum of such a preparation is, however, comparativelynarrow, since the effective and the thrombogenic dose are closelyadjacent, which could, e.g., be demonstrated in rabbits in the Wesslermodel, since in particular phospholipids constitute an increasedthrombogenicity risk.

The present invention thus has as its object to avoid the disadvantagesof the methods described and to provide a therapy principle for thetreatment of blood clotting disorders, in particular for the treatmentof factor VIII inhibitor patients, which enables, i.a., a simpleadministration, an effective onset of action, an increased half-life andthe avoiding of thrombogenic side effects.

According to the invention, this object is achieved with apharmaceutical preparation for the treatment of blood coagulationdisorders comprising, as active components, at least two coagulationfactors which are components of a prothrombinase or of apro-prothrombinase, respectively, in particular with a preparationcomprising, as active components, purified prothrombin and purifiedfactor Xa, in the solubilized state, one of the factors besidesprothrombin preferably being activated. Preferably, the components arepurified at least so far as to be free from endogenous, i.e.starting-material-derived, phospholipids, but also in particularphospholipid vesicles.

Thus, on the one hand, a premature thrombin formation is prevented andthe stability of the pharmaceutical preparation is ensured, and, on theother hand, the risk of thromboembolic side effects is minimized.

According to the invention, a mixture or a complex of at least twocomponents of the prothrombinase is understood as a "partialprothrombinase".

Beside the components of the prothrombinase or of thepro-prothrombinase, respectively, advantageously further factors ofblood coagulation and fibrinolysis are contained so as to obtain anattenuated effect, in particular an increase, decrease, acceleration ordeceleration of hemostasis. Accordingly, activators or pro-activators ofblood coagulation, among them factors of intrinsic or extrinsic bloodcoagulation, can further be contained as zymogens or as activatedfactors, as well as their agonists or antagonists or inhibitors.Besides, also the corresponding combinations are possible aspreparations which are administered separately. Among them are thecombination with fibrinogen which above all is suitable for localapplication.

According to a preferred embodiment, however, the pharmaceuticalpreparation substantially consists of the "partial prothrombinase", thecomponents of prothrombinase or of pro-prothrombinase, respectively,preferably as a complex. This complex can be purified and treated in asimple manner, in particular treated chemically and/or physically toinactivate molecular, microbial or viral pathogens.

The factors of the pharmaceutical preparations according to theinvention are contained in a form which enables activation of at leastone factor, or in which at least one factor has already been activated.As factors, preferably human factors are used. The factors are containedin the pharmaceutical preparations according to the invention, which arepreferably selected from the group consisting of the factors II, V, Va,X and Xa.

As inventive preparation of a partial prothrombinase orpro-prothrombinase, respectively, preferably combinations of factors IIand V or Va, respectively, as well as X and V or Va, respectively, areprovided. There, it is particularly preferred that the preparationaccording to the invention substantially consists of these combinations.Likewise, a pro-prothrombinase comprised of factors II and X, optionallyin combination with factor V or Va, respectively, is a preferredembodiment of the present invention.

Therein, native factors can be used, e.g. proteins or their equivalentsrecovered from plasma or from a plasma fraction, which are, e.g.,encoded by recombinant nucleic acids. Furthermore, also respectivederivatives which comprise the modified proteins or fragments are alsosuitable as long as they are activatable or have the respective activityto modulate the generation of thrombin.

The preparation according to the invention has the advantage that,despite its high stability in vitro, it is also stable in vivo until itsefficacy is shown by the activation of prothrombin and the generation ofthrombin at the site of the wound or of the bleeding. By the contactwith the cellular components of the vascular system, e.g. blood cellsand vessel walls, in particular phospholipid-containing surfaces,thrombin is generated in situ and hemostasis is promoted. On account ofthe local efficacy, systemic side effects, such as, e.g., thromboemboliccomplications, are avoided.

To effect a controlled influence on hemostasis, it is preferred to usehighly purified factors which have been purified from interferingcontaminants, in particular from a thrombin activity. The factors areespecially suitable which have been purified by chromatographic methods,such as ion exchange chromatography, hydrophobic chromatography,affinity chromatography and/or molecular exclusion chromatography.Thereby, specific activities of at least 50% of the theoretic purity, inparticular at least 70%, preferably at least 90% up to theoretic puritycan be attained for the individual factor. Accordingly it is alsopreferred to use factors which are substantially free from and devoid ofdenaturing products and thus are present as purified active factors,enzymes or as activatable zymogens, respectively.

Furthermore, it is preferred to carry out a treatment for theinactivation of infectious pathogens, e.g. by a treatment with chemicalsand/or by a physical treatment, such as a heat treatment, radiation orfiltration, in particular nanofiltration. According to a preferredvariant, the factors of the pharmaceutical preparation according to theinvention are treated with detergents, which, on the one hand, leads tothe inactivation of viruses and, on the other hand, solubilizes anyphospholipids possibly present.

Phospholipids may be contained in preparations of blood coagulationfactors, e.g. from plasma or from a plasma fraction or from a cellculture, respectively. The special treatment of the factors forseparation of the naturally present phospholipids comprises thesolubilisation thereof, on the one hand, and the separation of thephospolipids by the purification methods mentioned above, on the otherhand.

Although the preparation according to an embodiment may be utilized incombination with exogenic phospholipids, the preparations according tothe invention are preferably free from added phospholipids and containless than 0.01 mg of phospholipids/U prothrombin. Due to the possiblethrombogenic effect of phospholipids, the thrombogenicity risk isfurther markedly reduced. According to a particularly preferredembodiment, the preparations are free from detectable phospholipid.

According to a further embodiment, the preparation according to theinvention furthermore contains magnesium ions. These ions actcompetitively to calcium ions and can displace the calcium ions in theprothrombinase or pro-prothrombinase, respectively. Thus, a prematurethrombin formation in a solution of the preparation according to theinvention is prevented and the preparation thus is stabilized to anextent that it remains stable in a solution even for hours.

It has been shown that the pharmaceutical preparation may even beprovided as a stable infusion solution, primarily if it is ensured thatit does not contain any free calcium ions. For complexing the calciumions, also a content of a pharmaceutically acceptable chelating agent,e.g. EDTA, and related structures, such as citrate, are suitable.

The preparation according to the invention comprises a biologicalefficacy in animal models which is comparable to FEIBA® and it canmarkedly reduce the coagulation time of a factor VIII inhibitor plasma.It can completely normalize the prolonged bleeding time and intensity ofbleeding of factor VIII inhibitor rabbits and of von Willebrand factorinhibitor rabbits. By providing purified blood coagulation factors, e.g.purified prothrombin and purified factor Xa, the toxicity of thepreparation according to the invention is clearly reduced as compared toFEIB®. Thus, e.g., the effective combination of factor Xa andprothrombin proved negative in the Wessler-thrombosis model (J. Appl.Phys. 14 (1959), 943-946), i.e. for this combination, no thrombogeniceffects could be detected in the Wessler model even at higher doses ascompared to activated prothrombin complex.

In this thrombogenicity model, rabbits are anesthesized withpentobarbital, whereupon, under an additiional local anesthesia, thevena jugularis is prepared and provided with loose ligatures at adistance of 2 cm. Finally, the substance to be tested is injected within15 seconds into the ear vein opposite the vena jugularis. After further25 seconds, the ligatures are closed, and after a waiting time of 10 minthe ligatured vein section can be withdrawn and dissected in a Petridish filled with citrated buffer and evaluated. The evaluation criteria,modified according to Wessler, are as follows: no formation ofthrombi=0, a few small thrombi=0.5-1, a few, medium-sized and many smallthrombi=2, many medium-sized thrombi=3, a few large thrombi=3.5, onecoherent thrombus=4.

The components of the pharmaceutical preparation according to theinvention preferably have been purified to such a purity that even at adose of at least 150 U prothrombin/kg it is free from thromboembolicside effects, expressed by a score in the Wessler thrombosis model of 3at the most, preferably 2 at the most, in particular less than 2.

In the combination preparation of the invention, prothrombin iscontained preferably at a specific activity of at least 5 U/mg protein,more preferred at least 6, in particular at least 7, corresponding to50, 60 or 70% of the theoretic purity. The factor X or Xa preparationused should preferably have a specific activity of at least 100 U/mgprotein, factor Xa preferably being mainly contained as factor Xaβ.Factor V or Va, respectively, is used as co-factor at an approximatelyequimolar ratio to the coagulation factor of the partial prothrombinaseor pro-prothrombinase, respectively. Preferably, the ratio is (0.01-2):1(mol/mol), most preferably (0.5-2):1.

The preparation used should be as free from thrombin as possible, and itshould be possible to detect this freedom from thrombin by suitable,preferably chromogenic, tests (e.g. with the chromogenic substrate TH-1from IMMUNO AG).

It has been shown that, if the coagulation factors, such as, e.g.,prothrombin and factor Xa, are present as a complex in the preparationaccording to the invention, the preparation has an increased stabilityas compared to conventional preparations, and the complex furthermorecan be subjected to a further treatment for purification and/orinactivation of viruses. In a preferred embodiment of the preparationaccording to the invention, the preparation further comprisesantithrombin III in stabilizing amounts, optionally together withheparin. It is possible to demonstrate also such a preparation's freedomfrom thromboembolic side effects according to the Wessler test in theabsence or also after unmasking of the heparin, i.e. neutralisationand/or separation of the heparin, in a sense that the score of 3 willnot be reached.

A complex consisting of at least two coagulation factors which arecomponents of a prothrombinase or of a pro-prothrombinase, respectively,in particular in highly purified form, is of basic importance as a"partial prothrombinase complex". By the presence of calcium ions, suchas calcium chloride, the activity of a preparation on the basis of thiscomplex can be accelerated by a multiple. It has also been shown that apreparation comprising this complex and further calcium ions can be usedfor the preparation of a reagent for diagnostic purposes. A reagentwhich further comprises thrombin activity and optionally phospholipidsis, e.g., suitable to determine factor V cofactor activity. A diagnosticmethod using this reagent with or without activated protein C, aproteolytic inactivator of factor V, furthermore enables estimation ofan extent of an inactivation of factor V or a mutation-caused resistanceto activated protein C.

Surprisingly, within the scope of the present invention it has also beenfound that a pharmaceutical preparation comprising purified prothrombinas the only active component has an effect in vivo comparable to thecombination preparation of the invention in the treatment of coagulationdisorders, although in vitro with prothrombin alone no shortening of thecoagulation time of a factor VIII inhibitor plasma could be achieved.

Thus, a medical indication besides hereditary prothrombin deficiency fora pharmaceutical preparation comprising prothrombin as the only activecomponent has for the first time been shown.

The invention thus also relates to a pharmaceutical preparation, inparticular for the treatment of coagulation disorders, comprisingpurified prothrombin as the only active component.

Preferably, the prothrombin has been purified to such a degree that evenat a dose of at least 150 U prothrombin/kg, it is free fromthromboembolic side effects, expressed by a score of 3 at the most,preferably 2 at the most, in particular less than 2, in the Wesslerthrombosis model.

The combination preparation according to the invention and theprothrombin preparation, respectively, preferably comprise less than 0.1U of factor VIII:C or of factor VIII:Ag/U of prothrombin, and less than0.1 U of factor IX/U of prothrombin or less than 0.1 U of factor X/U ofprothrombin, respectively. By this, the undesired formation of orreaction with antibodies to these proteins can efficiently be prevented,and the risk of side effects can be reduced.

Although the prothrombin preparation can be used in combination withphospholipids, according to a further preferred variant of thepreparations according to the invention, these are free from addedphospholipids and contain less than 0.01 mg of phospholipids/U ofprothrombin. According to a particularly preferred embodiment, theprothrombin preparations according to the invention are free fromdetectable phospholipid.

The dosage of the preparations according to the invention is based onthe dosage of the equivalent components in FEIBA®. The Factor EightInhibitor Bypassing Activity (FEIBA) is defined as that activity of sucha preparation which reduces the coagulation time of a factor VIIIinhibitor plasma to 50% of the blank value in a clotting test asdescribed in AT 350726. On account of the high purity of the components,the advantages of the preparation according to the invention as comparedto FEIBA® reside in a reduced load of plasma proteins on the patient. Inparticular, the absence of factor VIII:Ag excludes the anaphylactic sideeffect. Thus it is possible to administer the preparations of theinvention at a concentration which provides for a dose which, e.g.,comprises at least 50 U prothrombin/kg body weight, this dose for thefirst time for such preparations being administrable even in a bolusinjection because of its freedom from side effects, and thus theotherwise time-consuming administration of such high doses as aninfusion can be avoided.

Usually the preparations according to the invention are administered ata dose of e.g. from 50 to 150 U prothrombin/kg body weight, the maximumdoses, however, possibly lying far beyond these 150 U/kg body weight(e.g., up to 300 or up to 500, respectively) without possiblethromboembolic side effects.

Thus, the invention also relates to administration forms of thepharmaceutical preparations of the invention which comprise a dose of atleast 50 U prothrombin/kg body weight, preferably of between 50 and 500U/kg body weight.

These administration forms may be ampoules or syringes which are alreadyprovided for an immediate application, or similar directly or indirectlyapplicable forms. Among them are containers which are suitable forinfusion, intramuscular or subcutaneous application or sets consistingof a container comprising the active agents as a lyophilisate and acontainer comprising a pharmaceutically acceptable solution suitable forreconstitution of the lyophilisate. Usually, the pharmaceuticallyacceptable solution or the pharmaceutical preparation, respectively,contains salts, preservatives, buffers and the like in an aqueoussolution (cf. Remington's Pharmaceutical Sciences, 15th edition, Easton:Mack Publishing Co., pp. 1405-1412 and 1461-1487 (1975), and TheNational Formulary XIV., 14th edition, Washington: AmericanPharmaceutical Association (1975)). Examples of non-aqueous solutionsare propylene glycol, polyethylene glycol, vegetable oils and injectableorganic esters, such as ethyl oleate. Aqueous carriers are, e.g., water,optionally mixed with alcohol, salt solutions (NaCl), Ringer's dextroseetc. As the preservatives, antimicrobial substances, antioxidants,chelating agents or inert gases may be used.

The preparations according to the invention are also suitable for localtreatment, application forms being chosen which become active at thesite of a bleeding. Among them are solids or liquids, preferably in theform of a powder, an adhesive tape or a wound cover, respectively,ointments, suppositories, capsules, in particulargastric-juice-resistant capsules, but also drops or sprays.

The coagulation factors used may be both of plasmatic origin andproteins prepared by recombinant DNA technology. What is essential inboth instances is that they are present in the pharmaceuticalpreparation in purified form, in particular in a form freed fromendogenous and exogenic phospholipids. Preferably, the pharmaceuticalpreparations according to the invention are provided in lyophilized formoffering the known transportation, storage and application advantages.As the reconstitution solution, a pharmaceutically acceptable solutionwhich optionally comprises ATIII or heparin, respectively, is suitable.Due to the high degree of purity of the components of the preparationsaccording to the invention, they can be reconstituted to an opticallyclear solution after a short reconstitution time at room temperature,preferably less than 5 min, in particular less than 1 min, with e.g. atleast 10 U prothrombin/ml, concentrations of even up to 200 Uprothrombin/ml solution being attainable. An optically clear solution isdefined by a maximum of the absorbance at 600 nm of 0.1 (for a solutionhaving a protein content of at least 5% by weight, at a layer thicknessof 1 cm), based on the pure (buffer) solution as a reference.Alternatively, also a solution having less than 70 light scatteringunits (LSU), determined by measurement in the nephelometer at 340 nm anda layer thickness of 1 cm is considered to be clear.

In contrast to the hitherto known preparations for treating bloodclotting disorders, the preparations according to the invention areextremely stable, i.e. they can be allowed to stand for a longer periodof time before being administered. For example, according to the productinformation, FEIBA® in its ready-to-administer state should not be leftto stand for more than 1 hour, whereas the preparations according to theinvention as a ready-to-use solution do not exhibit any clottingactivation or thrombogenicity even for a period of 3 hours or more atroom temperature, and thus the preparations according to the inventionmay also be provided as infusion solutions which can be administeredover a period of several hours. For the same reasons it is also possibleto administer the preparations of the invention over an extended periodof time as infusion solutions. It has been shown, however, as regardsthe thromboembolic side effects, there is no substantial differencebetween a bolus injection and a slow infusion with the preparationsaccording to the invention.

According to a preferred embodiment of the present invention, thepharmaceutical preparations are made available in suitable applicationdevices, preferably as a lyophilisate in syringes which allow for an insitu reconstitution with a pharmaceutically acceptable solution. For thecombination preparation an application device is suitable which is shownin FIG. 1, wherein the lyophilisates of prothrombin and factor Xa, e.g.,are preferably separately stored and can be administered when requiredafter an in situ reconstitution by means of a double-chamber syringe.

With the pure prothrombin preparation it suffices to provide theprothrombin in a simple syringe, preferably in the lyophilized state,optionally with a pharmaceutically acceptable solution forreconstitution (cf. FIG. 2).

The prothrombin preparation may, however, also be provided as a liquidpreparation or in liquid-deep frozen form, respectively.

Particularly if they are recovered from plasmatic proteins or from cellcultures, the pharmaceutical preparations according to the presentinvention may be subjected to one or several virus inactivationtreatments or treatments for virus depletion, respectively, e.g. to achemical or a chemical-physical treatment, to a heat and/or a detergenttreatment according to EP 0 159 311, EP 0 519 901 or EP 0 674 531, or toa physical treatment, such as nanofiltration.

The preparations according to the invention anable a safe and simpletreatment of blood coagulation disorders, in which an effective onset ofaction can be observed within a very short time.

It has been shown that the effective onset of action occurs more rapidlywhen administering the complex of factor II and factor Xa than with apreparation containing factor II exclusively. To treat a bleedingcomplication in a patient suffering from inhibitor hemophilia it is,thus, advantageous to treat the bleeding initially by administering thefactor II/Xa complex and thus attain a rapid hemostasis and to continuethe therapy with maintenance doses of a preparation containing factor IIonly, to avoid further bleedings.

Furthermore, the long half-life of the preparations according to theinvention and their freedom from thrombogenic side effects or theabsence of an anaphylactic reaction, which causes an increase in theinhibitor titer, enable a substantially improved treatment of thepatient affected with a risk of blood coagulation disorders as comparedto known methods. On account of the high concentration or dose of thepreparation according to the invention, the patient may receive a depotof the active substance which reduces the need of frequent treatments.The patient may also remain without treatment for a longer period ofseveral days and optionally may treat himself like an outpatient byself-injection, optionally subcutaneously.

The invention further relates to a method of preparing a purifiedpharmaceutical prothrombin preparation which is characterized in that aprothrombin complex concentrate is subjected to chromatographicpurification and the prothrombin-containing fraction is processed to apharmaceutical preparation. With the method according to the invention aprothrombin preparation is provided which fully meets all therequirements in respect of purity for the pharmaceutical preparations ofthe invention, in particular as regards the freedom from side effectsdetermined by means of the Wessler test.

In particular, the method according to the invention is characterised bythe combination of the following steps:

providing a prothrombin complex preparation in the solid state or as aprothrombin-containing solution,

virus inactivation treatment, preferably by heat treatment, inparticular in the solid state,

optionally dissolving the prothrombin complex preparation, therebyobtaining a prothrombin-containing solution,

optionally treating the prothrombin-containing solution with an alkalineearth salt as the solid carrier, prothrombin being adsorbed andsubsequently desorbed,

optionally concentrating one or several times, preferably byprecipitation or ultra/diafiltration, and gel filtration of theprothrombin-containing solution,

treating the prothrombin-containing solution with an anion exchanger,the prothrombin being adsorbed and subsequently selectively desorbed,

treating the prothrombin-containing solution with a hydrophobicchromatography material, and

finishing the prothrombin-containing solution into a pharmaceuticalpreparation.

Preferably, calcium phosphate, barium sulphate or aluminum hydroxide canbe used as the alkaline earth salt. In principle, all anion exchangerswhich have an affinity to prothormbin can be used as anion exchangers,such as, e.g., DEAE-Sephacel®, DEAE-Sephadext®, DEAE-Sepharose CL6B®,DEAE-Sepharose Fast Flow®, QAE-Sephadex®, Q-Sepharose Fast Flow®,Q-Sepharose High Performance®, Q-Sepharose Big Beads® (all fromPharmacia), DEAE-Tris-Acryl®, DEAE-Spherodexl®, Q-Hyper-D® (all fromSepracor); Macroprep DEAE®, Macroprep Q® (all from BioRad);DEAE-Toyopearl®, QAE-Toyopearl®, Toyopearl Super-Q® (all from Tosohaas),Protein PAK DEAE® (Waters); Fractogel EMD-TMAE®, Fractogel EMD-DEAE®,Fractogel EMD-DMAE®, Licrospher 1000 TMAE®, Licrospher 1000 DEAE® andLicrospher 4000 DMAE® (all from MERCK).

As the gel for the hydrophobic interaction chromatography, preferablyPhenyl-Sepharose High Performance® (Pharmacia), yet also otherchromatography gels, such as, e.g., Butyl-Sepharose®, Octyl-Sepharose®,Phenyl-Sepharose®, Phenyl-Sepharose Fast Flow High Sub®,Phenyl-Sepharose Fast Flow Low Sub® (all from Pharmacia), FractogelTSK-Butyl® (MERCK), Macroprep-Methyl-HIC-Support®, Macroprept-Butyl-HIC-Support® (all from BioRad); TSK-Gel Butyl Toyopear®, TSK-GelPhenyl Toyopearl® and TSK-Gel Ether Toyopearl® (all from Tosohaas) areused.

A possible embodiment of the method according to the invention isillustrated in FIG. 3.

The prothrombin preparation which can be prepared by the methodaccording to the invention is not only characterised by an extremelyhigh purity which is close to the theoretically possible purity of 10U/mg, but also by the fact that even at a dosis of at least 150 U ofprothrombin/kg it is free from thromboembolic side effects, expressed bya score in the Wessler thrombisis model of 3 at the most, preferably 2at the most, in particular less than 2, and therebeyond that as alyophilisate it can be reconstituted with a reconstitution time of 1 minat the most to a clear solution having an activity of at least 10 U ofprothrombin/ml up to 200 U of prothrombin/ml. The biological activity ofthe prothrombin preparation is to be understood as the enzymaticactivity obtained upon activation of the prothrombin.

According to a further aspect, the present invention relates to the useof purified prothrombinase factors, in particular of purifiedprothrombin and, optionally, purified factor Xa, for producing apharmaceutical preparation for establishing supranormal prothrombinconcentrations in a patient's blood, or to establish normal prothrombinconcentrations in blood in case of conditions involving a loweredprothrombin level.

It has been shown that with the preparations according to the inventionsuch supranormal factor II levels can be maintained even permanently,i.e. over extended periods of time, which, on the one hand, goes back tothe fact that for blood coagulation factors, prothrombin has a very highhalf-life as a medicament, and, on the other hand, also resides in thefact that the preparations according to the invention are free from sideeffects so that even a subcutaneous administration, e.g. by depotadministration, is possible. The supranormal concentrations ofprothrombin which are possible in blood by the administration ofpurified prothrombin and optionally purified factor Xa or otherprothrombinase factors, amount to at least 150%, preferably even atleast 200%, corresponding to an activity of at least 1.5 U ofprothrombin/ml blood, preferably at least 2.0 to up to 10 U/ml.

Finally, the invention relates also to the use of purifiedprothrombinase factors, in particular of purified prothrombin and,optionally, purified factor Xa, for preparing a pharmaceuticalpreparation for the treatment of factor VIII inhibitor conditions,hemophilia A or B, and of von Willebrand Disease. It has been shown thatin the animal model, with the preparations according to the invention arapid and efficient treatment which is free from side effects ispossible for all these indications.

Preferably, the preparations according to the invention are provided ina solution having a physiological pH, which preferably does not containany free calcium ions. However, it is also possible to use an acidbuffer in the pH range of from 4.5-6.5, preferably 5-6, which is removedfrom the factor Xa activity optimum, by which the activation ofprothrombin is avoided and therefore the stability of the combinationpreparation may once more be increased. It goes without saying that allthe pharmaceutical additives and solutions suitable for factors II, V,Va, X and Xa may be used for the ready-to-use production of thepreparations according to the invention.

Furthermore, it has surprisingly been shown that with the preparationsaccording to the invention even in non-hemophiliacs, acute bleedings, anincreased bleeding intensity or an increased risk of bleeding mayeffectively be treated. In addition to the above-mentioned indicationsof the various types of hemophilia, i.e. hemophilia A and B as well asinhibitor hemophilia, the preparations according to the invention arealso applicable in non-hemophiliacs. Among the non-hemophiliacs thereare also those who have blood coagulation disorders based on inhibitorsagainst blood factors which are not factor VIII or factor IX.Furthermore, patients can be treated who exhibit an impaired thrombingeneration, which is caused by the absence or functional defect of oneor several factors of the extrinsic or intrinsic coagulation or at theformation of antibodies to one or several of these factors or by a lackof the cellular receptor for one or several of these factors. Uponadministration of a preparation according to the invention, acoagulation-promoting effect optionally may occur in vivo, whereby atreatment, i.e. a prophylactic or therapeutic administration becomesfeasible.

According to a further aspect, the present invention thus relates thethe use of at least 2 coagulation factors which are components of aprothrombinase or of a pro-prothrombinase, or to the use of thepreparation according to the invention for preparing a pharmaceuticalpreparation for the treatment of acute bleedings, an increased bleedinginsensity or an increased risk of bleeding in non-hemophiliacs.

In particular, conditions can be treated which are caused by an impairedaggregation behavior of blood platelets or thrombopathies, e.g.storage-pool defects, or due to a deficient or dysfunction ofplatelet-associated proteins, but also bleeding conditions caused byplateles deficiency (thrombocytopenia). One side effect of ananticoagulant therapy resides in the heparin-induced thrombocytopenia,which also constitutes an indication for the preparation acording to theinvention.

According to a preferred embodiment, the present invention thus relatesto the use of at least 2 coagulation factors which are components of aprothrombinase or of a pro-prothrombinase, respectively, for preparing apharmaceutical preparation for the treatment of bleedings caused by athrombocytopenia, in particular for the treatment of a heparin-inducedthormbocytopenia.

In this connection it is advantageous that the pharmaceuticalpreparation prepared according to the method of the invention may have aprimary hemostatic activity. The efficacy of the pharmaceuticalpreparation prepared may be further enhanced by the combination withproteins having primary hemostatic activity. Primarily suited is a vonWillebrand factor protein or a fraction of the von Willebrand factor,respectively, with a defined collagen binding activity.

A further indication for the treatment of patients suffering from bloodcoagulation disorders is the prevention or treatment of bleedingsoccurring in connection with von Willebrand Disease. This disease withor without a prevalence of coagulation factor inhibitors involves anincreased bleeding propensity or a risk of bleeding and in many cases itleads to bleedings which are difficult to control. By aid of thepreparation according to the invention it is possible to quickly stopsuch a bleeding.

Thus, according ot the invention, a set for the treatment of patientssuffering from blood coagulation disorders is provided, which comprisesthe following components:

a) a pharmaceutical preparation comprising at least 2 coagulationfactors which are components of a prothrombinase or of apro-prothrombinase and which are free from phospholipids, and

b) a protein having primary hemostatic activity, in particular vWF.

Bleedings which are difficult to control may in some instances occur asa side effect of a therapy with synthetic, semi-synthetic and biologicalcoagulation inhibitors or anticoagulants or thrombocyte functioninhibitors. These substances interfere directly or indirectly with thecoagulation system and may disturb the natural coagulation or clottingprocess in an undesired way. Thus, there is a demand for antagonists forthese substances. In the prior art, the use of a prothrombin complex orof a FEIBA® preparation is known for the treatment of bleedings whichhave been caused by an anticoagulant therapy. In this respect, cf. IraniM. S. et al., The American Journal of Cardiology, Vol. 75, Feb. 15,1995, p. 422; Fareed J. et al., Haemostasis 1991, Vol. 21 (suppl. 1),pp. 64-72; and Fareed J. et al., Seminars in Thrombosis and Hemostasis1991, Vol. 17, No. 2, p. 137.

According to a further embodiment of the present invention, theabove-stated coagulation factors are used for preparing a pharmaceuticalpreparation according to the invention for the treatment of bleedingswithin the scope of an anticoagulant therapy, in particular forpreparing a pharmaceutical preparation as an antidote for a coagulationinhibitor or for an anticoagulant or for a thrombocyte functioninhibitor. What is substantial is that a risk of thrombosis involvedwith the presence of phospholipids is avoided. Particularly withpatients who are subjected to a therapy with anticoagulants, there is anincreased risk of thrombosis. According to the invention, however, theuse of phospholipids can be obviated, whereby surprisingly the antidoteeffect becomes even more specific.

The use according to the invention primarily relates to the antagonizingof a coagulation inhibitor or anticoagulant which indirectly or directlyinhibit factor Xa or thrombin. According to the invention, preferably asubstance is antagonized which is selected from the group consisting ofAPAP((2S)-2-[4-[[(3S)-1-acetimidyl-3-pyrrolidinyl]-oxy]-phenyl]-3-(7-amidino-2-naphthyl)-propionic acid hydrochloride pentahydrate), benzamidine derivative,hirudin, heparin, heparin analogues, in particular pentasaccharides, ATIII heparin complex, AT III, antistasin, "Tick-Anticoagulant Peptide",inactive coagulation factors, in particular "active site"-inhibitedcoagulation factors, TFPI, competitive ligands for thrombocyte membranesurface receptors, in particular antibodies to GP IIb/IIIa, and theirgenetically engineered or synthetically produced analogues, inparticular peptides, as well as oral anticoagulants. Among the inactivecoagulation factors there are primarily derivatised, mutated,fragmented, chemically or physically inactivated or inhibited factors ofintrinsic and extrinsic blood coagulation, respectively, which maycompetitively interact with the native factor.

The antagonizing of a thrombocyte-function inhibitor is primarilyindicated in the case of ticlopidin or acetyl salicylic acid.

Acute bleedings are primarily extremely critical in the region of thebrain. Therefore, there is a need for the prevention or treatment,respectively, of intracranial bleedings, e.g. intraventricularhemorrhage (IVH). Particularly patients suffering from damaged bloodvessels or from an impaired thrombin generation run an increased risk ofintracranial bleedings. The preparation according to the ivnention isalso specifically suitable for this indication, wherein above all theimproved treatment of premature infants becomes possible.

Within the scope of the use according to the invention at theindications listed, the respective kits are each provided which comprisethe pharmaceutical preparation of the invention as one component.Furthermore, substances which intensify the hemostatic effect, e.g. aprotein having primary hemostatic activity, in particular vWF, may becontained. Besides this, a kit for anticoagulant therapy naturally willcontain the respective anticoagulant and the pharmaceutical preparationof the invention as an antidote.

The invention will now be explained in more detail by way of thefollowing Examples and with reference to the associated drawing figures,to which, however, it is not to be restricted.

Therein,

FIGS. 1 and 2 show possible forms of adminstration of the preparationsaccording to the invention;

FIG. 3 shows the flow diagram of one embodiment of the preparationmethod;

FIG. 4 shows the demonstration of the partial prothrombinase complex;

FIG. 5 shows the spectroscopic analysis of one example of thepreparation (A) according to the invention as compared to standardpreparations [activated prothrombin complex (B), prothrombin complex(C)];

FIG. 6 shows the in vivo effect of a factor II preparation on factorVIII inhibitor rabbits;

FIGS. 7 and 8 show the in vivo effect of the preparations according tothe invention in the von Willebrand factor/factor VIII inhibitor model.

EXAMPLE 1

Preparation of factor X and factor II from a virus-inactivated plasmafraction by means of ion exchange chromatography.

A lyophilized prothrombin complex factor preparation containing factorsII, IX, X as well as protein C and protein S was prepared according tothe method of Brummelhuis, H. G. J., Preparation of the Prothrombincomplex, in Methods of Plasma Protein Fractionation, Curling, J. M. ed.,117-128, Academic Press, New York, (1980), and heat-treated for virusinactivation according to EP 159 311. Accordingly, the lyophilisate(1,000 U of factor X/g, 1,200 U of factor II/g) was dissolved indistilled water so that the latter contained 50,000 U of factor X/l, andadjusted to pH 7.0. After the addition of 12% (v/v) TWEEN® 80 it wasstirred for 1 hour at room temperature. Subsequently, it was diluted 1:5with a 20 mM Tris-HCl-buffer, pH 7.0, and the prothrombin complexprotein fraction was adsorbed on calcium phosphate [Ca₃ (PO₄)₂ ] at aconcentration of 30 g Ca₃ (PO₄)₂ per 1 of prothrombin complex solutionby stirring for one hour at room temperature. Subsequently, the solidphase was separated by centrifugation at 5,000 rpm for 20 min, and theprecipitate was washed twice with 20 mM Tris-HCl-buffer, pH 7.0,containing 10% ammonium sulphate, by resuspension and renewedcentrifugation. A third washing was performed in an analogous mannerwith 20 mM Tris-HCl-buffer, pH 7.0, containing 150 mM NaCl. Elution ofthe prothrombin complex fraction was performed with 1 M sodium phosphatesolution, pH 7.0, wherein 25 ml of this solution per g of calciumphosphate were stirred for 1 hour at room temperature and subsequentlythe remaining precipitate was separated by centrifugation as above.Subsequently, the supernatant was subjected to an ammonium sulphateprecipitation with 366 g of ammonium sulphate per 1 for 15 h at 4° C.under stirring. The precipitate which contained the prothrombin complexfraction was centrifuged off as above. The precipitate was taken up in a25 mM trisodium citrate dihydrate buffer, containing 100 mM NaCl, 1 mMbenzamidine hydrochloride, pH 6.0, and re-buffered on a column filledwith Sephadex® G-25 at 4° C. with a linear flow of 1 cm/min against 25mM trisodium citrate dihydrate buffer, containing 100 mM NaCl and 1 mMbenzamidine hydrochloride, pH 6.0, to separate the ammonium sulfate.Therein, UV absorption at 280 nm and electric conductivity were measuredin the eluate flow. The protein-containing fractions were combined andsubsequently subjected to an ion exchange chromatography overDEAE-Sepharose FF® (Pharmacia). The fractions were applied to a column(inner diameter:gel bed height=1:1.3) having a gel volume of 8.2 1, 0.55g of protein/l gel, at a linear flow of 0.36 cm/min. Chromatography wasperformed at 22° C. Prior to application of the proteins, the column hadbeen equilibrated with a 25 mM trisodium citrate dihydrate buffer,containing 100 mM NaCl, 1 mM benzamidine hydrochloride, pH 6.0. Elutionof the protein fraction was effected in several stages with a buffer 1(25 mM trisodium citrate dihydrate, 1 mM benzamidine hydrochloride, 245mM NaCl, pH 6.0), a buffer 2 (25 mM trisodium citrate dihydrate, 1 mMbenzamidine hydrochloride, 270 mM NaCl, pH 6.0), and a buffer 3 (25 mMtrisodium citrate dihydrate, 1 mM benzamidine hydrochloride, 400 mMNaCl, pH 6.0). Elution with buffer 1 was carried out with 2.4 columnvolumes, inert protein being separated therewith. Elution with buffer 2was carried out with 5.6 column volumes, and there fractions werecollected which were analysed for their contents of factor II, factor X,protein C and factor IX. The factor X-containing fractions which werefree from factor II, IX and protein C, were combined. By elution withbuffer 3 (1.9 column volumes), factor II was desorbed, wherein againfractions were collected and assayed for their contents of factor X,factor IX and factor II. The factor II-containing fractions were pooled.Both, factor II and the factor X-containing pool optionally could besubjected to an additional treatment for inactivation of pathogenicimpurities by the addition of 1 M KSCN and incubation at 22° C. forseveral hours.

EXAMPLE 2

Purification of factor II by means of hydrophobic interactionchromatography

The factor II pool recovered in Example 1 was adjusted to 1.8 M NaCl bythe addition of sodium chloride, and the pH was corrected to pH 7.0.Subsequently, this solution was adsorbed to a gel, Phenylsepharose HighPerformance® (Pharmacia), by hydrophobic interaction, wherein 3 g ofprotein/l gel were bound. In a column having a ratio of innerdiameter:gel bed height=1:1.9, the protein fraction was adsorbed at alinear flow of 0.25 cm/min, and subsequently freed from inert protein bywashing with a buffer (25 mM Tris-HCl, 3 M NaCl, pH 7.4). Factor II waseluted from the column by gradient elution with 11.5 column volumes of 3M-0.9 M NaCl with simultaneous collection of fractions, and thosefractions which contained factor II activity but were free from factor Xand factor IX, were pooled. Subsequently, the collected factor IIfractions were concentrated ten-fold by ultra/diafiltration via anultrafiltration membran having a cut-off of 30 kD, and re-bufferedagainst a buffer containing 4 g of trisodium citrate dihydrate/l, 8 gNaCl/l, pH 7.0. A thus prepared factor II preparation had a specificactivity of 6.9 U/mg protein. The determination of the factor IIactivity was effected with the 1-stage method based on thethromboplastin time, by using a factor II-deficient plasma against theinternational factor II standard by using the reagent combination fromIMMUNO, Vienna. In clotting analyses, other coagulation factors weredetectable in traces or not at all (factor VII<0.00002 U/U factor II,factor IX 0.0002 U/U factor II, factor X 0.004 U/U factor II, protein C0.003 U/U factor II, and factor VIII<0.0002 U/U factor II).

EXAMPLE 3

Purification of factor II by means of hydrophobic interactionchromatography and hydroxyl apatite chromatography

As an alternative preparation method for a highly purified factor IIalso a method was used, in which at first factor IX was separated from alyophilized prothrombin complex factor preparation (cf. Example 1) byhydrophobic chromatography, subsequently factor II was isolated, and thelatter was then highly purified by chromatography on hydroxyl apatite.The prothrombin complex factor preparation was dissolvd as in Exmaple 1and incubated with a detergent at room temperature for 1 h.Subsequently, a factor II, IX and X-containing fraction was isolated asin Example 1 by ion exchange chromatography on DEAE-Sepharose FF®(Pharmacia). From this, the factor IX-containing fraction wassubsequently removed by interaction with Butyl-Toyopearl® (Toso Haas).Subsequently, the adsorption supernatant was purified by a furtherhydrophobic interaction chormatography on Phenyl-Sepharose HighPerformance® (Pharmacia), wherein approximately 4 g of protein/l gelcould be adsorbed. In a column having a ratio of inner diamter:gel bedheight=1:1.9, the protein fraction was adsorbed at a linear flow of 0.25cm/min, subsequently the inert protein was removed by washing with 20 mMTris-HCl, 3 M NaCl, pH 7.4, and finally the factor II-containingfraction was isolated by step-wise elution which desorbed from gel withdecreasing conductivity at 1.9 M NaCl. The factor II-containing fractionwas then directly adsorbed on Ceramik-Hydroxylapatite® (Biorad). Thiswas carried out on a column having a ratio of inner diameter:gel bedheight=1:4.8. Elution was done at a linear flow of 3 cm/min. By elutionwith a salt gradient, factor II could be desorbed from the column. Thefactor II-containing fractions were collected and concentrated viaultra/diafiltration over polysulfon membranes with a cut-off of 30 kD,until the factor II concentration was 50-100 U/ml. A thus preparedfactor II preparation had a specific activity of at least 7 U/mgprotein. Other coagulation factors, in particular factor IX and factorVIII, were detectable only in traces or not at all, as in thepreparation from Example 2. By selection of a suitable diafiltrationbuffer, the factor II preparation was transferred into apharmaceutically compatible buffer (e.g. 4 g of trisodium citratedihydrate/l, 8 g NaCl/l, pH 7.0).

EXAMPLE 4

Recovery of factor Xa

The factor X fraction prepared as described in Example 1 subsequentlywas further processed to factor Xab as described in DE 43 25 872,wherein the thus-obtained highly purified factor Xa preparation waslyophilized in the presence of 1 g/100 ml human albumin. Such apreparation was free from other clotting factors; the contained factorXa had a specific activity of 120 U/mg protein before the addition toalbumin.

EXAMPLE 5

Lyophilization of factor II

The preparation containing highy purified factor II described in Example3 was lyophilized without the addition of stabilizers, more than 80% ofthe initial activity remaining after lyophilization.

EXAMPLE 6

Colyophilization of factor II and factor Xa

A factor II preparation prepared according to Example 3 was filled at aconcentration of 100 U/ml at 20 ml into 50 ml bottles and shock-frozenat -80° C. Subsequently, a solution of a highly purified factor Xa whichhad been prepared according to DE 43 25 852 and had a concentration of500 U/ml was dosed in an amount of 30 μl onto the frozen factor IIsolution. By immediate freezing of the small volume, a mixing of thefactor II and factor Xa phases was prevented. Subsequently, it waslyophilized. To prepare the infusion solution, the lyophilzate wasreconstituted with 20 ml A. dest., mixed, and immediately prepared foradministration.

EXAMPLE 7

Pharmaceutical formulation of factor II, factor Xa and antithrombin IIIand/or antithrombin III heparin complex

The highly purified factor II in combination with factor Xa andantithrombin III or antithrombin III heparin complex were diluted to theapplication concentration in a buffer containing 4 g of trisodiumcitrate dihydrate/l and 8 g NaCl/l, pH 7.0. These solutions could belyophilized, an activity of at least 80% of the respective componentsremaining.

EXAMPLE 8

Detection of partial prothrombinase

Formation of a complex of factor II and factor Xa to "partialprothrombinase" was demonstrated by the following experiment:

57 U of factor II from Example 3 and 1.2 U of factor Xa from Example 4were dissolved in 20 mM Tris-HCl buffer, containing 150 mM NaCl, pH 7.4,and incubated for 15 min at room temperature for the formation of thecomplex. Subsequently, one aliquot of the solution was chromatographedby gel permeation chromatography over Superose® 12 (HR10/30) (Pharmacia)at a flow rate of 0.25 ml/min. The application volume was 200 μl. Thethroughput through the column was measured UV-spectrophotometrically at280 nm and collected in fractions of 0.5 ml. Subsequently, factor Xawhich was determined with a chromogenic substrate in a photometric testby means of the method described in DE 43 25 872 was measuredquantitatively in the fractions. Likewise, the factor II activity wasdetermined in the individual fractions as in Example 2. The result canbe taken from FIG. 4. Factor Xa [FIG. 4: - -▪- - Act. Xa (FII/Xa)] andfactor II [FIG. 4: - -- - Act. II x 10(FII/Xa)] eluted together withthe protein fraction [FIG. 4: -- A280 nm (FII/Xa)]. Under identicalconditions, factor Xa then was applied alone to the column, the elutionprofile was determined after gel passage by measureing the UV absorptionat 280 nm [FIG. 4: .......A280 nM (FXa)] and factor Xa activity [FIG.4: - -▴- - Act.Xa×10 (FXa)] was determined in the fractions. The proteinpeak corresponding to factor Xa was clearly offset from factor Xa in thecomplex with factor II. By reducing the retention time of factor Xa inthe complex on the column and together therewith for displacement to anapparently higher molecular mass, it was possible to proove a complexformation of factor II and factor Xa (partial prothrombinase).

EXAMPLE 9

Formulation of the preparation in the double chamber syringe system

To simplify the application of the multiple-component system, such as,e.g., mixtures of factor II and factor Xa or factor II, factor Xa andantithrombin III, a double double-chamber syringe body, as described inAT 382 783, can be used as application device. In the clinicalapplication of lyophilized multiple-component systems, the latter wouldotherwise each have to be reconstituted and mixed with each other at adefined ratio before being administered to the patient. Filling of thecorresponding lyophilisate into a double-chamber syringe system allowsfor an exact dosing to a pre-determinable activity of the preparationfor the treatment of inhibitor hemophilia, e.g. to conventional FEIBAunits, which can be determined according to AT 350 726. By using such asystem, the effective mixture is prepared in situ at the injection. Inone embodiment, lyophilisates of the active substances, factor II andfactor Xa, are each present in the two double-chamber syringe bodies,which, on account of the ready solubility of the highly purifiedproteins, solubilize at once and are immediately infused into thepatient by further pressing down the syringe piston after mixing in themixing head (cf. FIG. 1).

On account of the high stability of factor II in solution, the formeralso is suitable as a solvent for factor Xa in the application form of adouble-chamber syringe. A solution of highly purified factor II,prepared e.g. according to Example 2, in a physiologically compatiblecitrate buffer (4 g of trisodium citrate dihydrate/l, 8 g NaCl/l, pH7.0) at a concentration of 100 U factor II/ml, is admixed withantithrombin III (IMMUNO, Vienna) (1 mU antithrombin III/U factor II).This solution is used as a solvent for the lyophilized powder of ahighly purified factor Xa in a double-chamber syringe (cf. FIG. 2).

EXAMPLE 10

Stability of the highly purified factor II

Factor II was purified as described in Example 2 and stored as asolution at a concentration of 60 U/ml in a buffer containing 4 g/ltrisodium citrate dihydrate, 8 g/l NaCl, pH 7.0, at 5° C., at 22° C., at37° C. and at 50° C. At the storage temperatures of 5° C. and 22° C.,samples were drawn every 24 h, at the storage temperatures of 37° C. and50° C., samples were taken over 24 h at 1 h, 2 h, 4 h, 8 h and 24 h. Ineach sample the factor II activity was determined.

At 5° C. more than 80% of the initial activity could be detected even 86days after storage had been started. At 22° C., more than 80% of theinitial activity was found for 14 days; at 37° C., 95% of the initialactivitiy could be found 24 h after storage had been started. Even at50° C., 94% of the original activity could still be found 8 h afterstorage had been started.

EXAMPLE 11

Autoactivation and stability of the preparations according to theinvention

Inventive preparations according to Example 5 (factor II) and Example 6(factor II/Xa complex) were examined in an in vitro-test for theirprothrombotic properties particularly of the extrinsic clotting system,as compared to two commercially available prothrombin complexconcentrates. Before being used in the test, the heparin present in theprothrombin complex concentrate was neutralized with protamin sulphatecorresponding to the concentration (of heparin) so as to exhibitthrombogenic substances that would be masked by heparin.

The analysis material consisted of 250 μl Thrombotest® (Nycomed PharmaAS, Oslo, Norway), a preparation containing thromboplastin from bovinebrain and adsorbed bovine plasma, which was pre-incubated for 3 min at37° C. Subsequently, 50 μl of a sample were added, and the clotting timewas determined by means of a spherical coagulometer (KC4, Amelung).Bovine thromboplastines are considered to be particularly sentitive toactivated clotting factors. Accordingly, the test system can provideinformation on the in vitro-thrombogenicity of the preparations assayed.In this test, undiluted normal human plasma was used as a control. Thelatter had a clotting time of 74 seconds. When used undiluted, the twoassayed preparations of the invention had clotting times of more than100 seconds, the concentration corresponding to a possible applicationconcentration of 30 U of factor II/ml. E commercially availableprothrombin complex concentrate, likewise dissolved in the applicationconcentration and diluted to 30 U/ml, had to be further diluted withbuffer 1: 32 to achieve the clotting time of normal plasma (74 seconds).A further commercially available, activated prothrombin complexconcentrate even had to be diluted with buffer 1: 216 so as to achievethe clotting time of normal plasma, i.e. 74 seconds. Subsequently, thepreparations according to the invention were stored in the dissolvedstate at room temperature for up to 4 h and samples were taken once perhour, and again the clotting time was determined by means ofThrombotest®. No change had taken place, i.e. even after 4 h the samplesused in the undiluted state still have clotting times of more than 100seconds. From these tests it could be seen that the preparationsaccording to the invention have a markedly lower thrombogenicitypotential as compared to commercially available prothrombin complexconcentrates.

EXAMPLE 12

Dissolution behavior of the preparation according to the invention

A highly purified factor II-containing preparation was prepared asdescribed in Example 5 and lyophilized. The lyophilisate was adjustedsuch that the volume activity was 50 U/ml after reconstitution withdistilled water. This was a typical application concentration. Onaccount of the high purity of the preparation, however, also volumeconcentrations of 100 and more units of factor II per ml could beachieved. The dissolution time of the preparation, defined as that timewhich passes from the addition of the solvent (distilled water) untilthe complete dissolution of the powder was determined. In comparisonthereto, the lyophilisates of two commercially available preparations,i.e. prothrombin complex and activated prothrombin complex, were eachreconstituted according to the supplier's instruction at the applicationconcentration by the addition of distilled water, and the dissolutiontimes were also determined. The dissolution times of the testedpreparations can be taken from the following table:

    ______________________________________                                                   Preparation of                                                                the invention                                                                             APCC.sup.1                                                                            PCC.sup.2                                      ______________________________________                                        Dissolution time (s)                                                                       <30           240     300                                        ______________________________________                                    

¹ APCC=activated prothrombin complex concentrate

² PCC=prothrombin complex concentrate

When the respective preparations had been dissolved, an UV/VIS spectrumbetween 280 and 750 nm was measured of each of them, against a citratedsaline buffer. The spectra can be taken from FIGS. 5a, b and c (a:preparation according to the invention; b: activated prothrombincomplex; c: prothrombin complex). A comparison of the specta showed thatthe preparation according to the invention had a markedly lower lightabsorption in the visible range up to 700 nm than the preparations ofcomparison. The preparation according to the invention thus was markedby an excellent solubility and, in the dissolved state, was present as aclear, uncolored solution, as is characteristic of an improvedpharmaceutical preparation.

EXAMPLE 13

Effect of the preparation in factor VIII inhibitor plasma

A high-titer factor VIII inhibitor plasma (55 BU/ml) was pre-cooled inan ice bath and incubated for one minute and at 37° C. with PTT reagent(IMMUNO, Vienna) and the sample to be tested, both also pre-cooled in anice bath, at a ratio of 1+1+1. Subsequently, 1 part of an 0.05 M CaCl₂solution was added, and the clotting time of the test mixture wasdetermined by means of a spherical coagulometer (Amelung), model KC-4.The preparations to be tested were further diluted 1:10 in a buffercomprising 7 g NaCl/l and 6 g of trisodium citrate dihydrate/l, and usedfor the test at this concentration. Under these conditions, highlypurified factor II, alone and in combination with highly purified factorXa, in combination with antithrombin III and heparin was tested. Theclotting times can be taken from the following table.

    ______________________________________                                        Test substance                                                                (concentration)            Clotting                                           Factor II                                                                             Factor Xa                                                                              Antithrombin III                                                                           Heparin                                                                              time (sec)                               ______________________________________                                        10 U/ml --       --           --     147                                      10 U/ml 0.1 U/ml --           --     63                                       10 U/ml 0.1 U/ml 1 U/ml       --     55                                       10 U/ml 0.1 U/ml 1 U/ml       6 U/ml 49                                       ______________________________________                                    

As the control, the clotting time of the test sample was determined withpure sodium chloride trisodium citrate buffer. The latter was 148seconds. Thus, the clotting time of the inhibitor plasma could bemarkedly reduced merely by the addition of factor II/Xa with or withoutantithrombin III or heparin. By measuring a FEIBA standard preparation(FEIBA STIM 4, IMMUNO), which is the conventional preparation for thetreatment of factor VIII inhibitor patients, at various concentrationsin the same test sample it could be found that the FEIB-activity of thepreparation containing 10 U/ml factor II and 0.1 U factor Xa/mlcorresponds to approximately 25 U FEIBA/ml.

EXAMPLE 14

In vivo-effect with factor VIII inhibitor hemophilia

To test the in vivo efficacy of the preparation according to theinvention, a factor VIII inhibitor hemophilia rabbit model was used.White New Zealand rabbits weighing approximately 2 kg each wereanesthesized. After the onset of anesthesia, each right femoral vein wasprepared and a permanent venous access was established. Through thelatter, per kg body weight, 0.5 ml of a human factor VIII inhibitorplasma (1500 BU/ml) were infused during 10 min. 30 min after the end ofinfusion, the bleeding characteristic was determined by using a modifiedmethod according to Giles et al., Blood 60:727-730 (1982). For this, thefur surrounding the claw of a hind paw of the rabbit was shaved toprevent blood emerging at the later bleeding from being absorbed by thefur. The nail cuticle was injured by means of a nail clipper;immediately thereafter, filters were placed therebelow such that theblood could drip directly onto the filter without being sucked upthereby on account of a capillary effect, so as to prevent a formingblood clot from being destroyed. The filter units were exchanged every 2minutes, and the emerging blood was collected in fractions. Blood wascontinued to be collected for 30 minutes, and then the wound wascauterized if the bleeding had not stopped. To quantitate the bleedingcharacteristic, the filters were each extracted with 0.04% ammoniumhydroxide solution for 5 h, whereby the erythrocites, which had beencollected in the filter with the blood, were lysed. By a 10 minsonication, the hemoglobin was extracted and quantitated photometricallyat 416 nm against a calibration curve. The latter was established bypipetting rabbit blood in volumes of between 10 μl and 1 ml ontofilters, extracting the filters as described above and photometricallydetermining the hemoglobin at 416 nm. The bleeding characteristics ofthe nail cutting was determined by graphically plotting the amounts ofblood per 2 min fraction against time. For an evaluation, theaccumulated blood loss was determined by graphically plotting the volumeof the individual blood fractions against time. The slope of thecumulative bleeding between 10 and 20 min was taken as a relevantbleeding criterion. This value was independent of the initial bloodamount which was subject to variations from rabbit to rabbit, dependingon the claw cut. The increase of the bleeding characteristic in 10 to 20min observation intervals served as a measure for the intensity ofbleeding. An increase equal zero meant that the bleeding had stopped, anincrease >0 with a correlation coefficient of >0.8 meant that there wasa constant bleeding. Under the test conditions, healthy rabbits had ableeding intensity of <2 μl blood/min. Factor VIII inhibitor rabbitsexhibited a bleeding intensity of approximately 50 μl blood/min (cf.FIG. 6). Factor VIII inhibitor rabbits which had been treated with afactor II preparation according to Example 3 at a dosage of 75 U/kg bodyweight as an infusion over 30 min showed a mean (n=6) bleeding intensityof 9.3 μl/min and thus a significant reduction as compared to theuntreated inhibitor animal.

EXAMPLE 15

In vivo effect in case of von Willebrand factor/factor VIII inhibitor

Analogous to Example 14, a von Willebrand factor/factor VIII inhibitormodel was established by infusing rabbits with an anti-von Willebrandfactor/factor VIII antiplasma from goat which had been recovered byimmunization of goats with a purified factor VIII/von Willebrand factorpreparation, at a dosage of 1 ml/kg body weight. Such pre-treatedanimals exhibited an increased intensity of bleeding (cf. FIG. 7). Thebleeding characteristics were measured by claw cuts performed on theseanimals, simultaneously with an infusion of the test substance and 30min after infusion of the test substance had ended. By administeringfactor II as a bolus of 2.5 ml at a dosage of 75 U/kg, the initiallyincreased bleeding intensity could be reduced from 77 μl/min to 31μl/min (1st claw cutting) and 12 μl/min (2nd claw cutting),respectively. By combining-factor II (75 U/kg) with a factor Xab(prepared according to Example 4) at a dosage of 0.55 U/kg as a bolus,at an injection volume of 2.5 ml, the propensity to bleeding could bereduced from the initial value to 18 μl/min at the first claw cuttingand 5 μl/min at the 2nd claw cutting. Thus, the abnormally increasedbleeding behavior of the inhibitor animals was normalilzed to the levelof the healthy control animals (4 μl/min).

In addition, also the combination of factor II (75 U/kg), factor Xa(0.55 U/kg) and antithrombin III (IMMUNO Vienna) (75 mU/kg) was examinedin the same model. At the first claw cut, a reduction to 35 μl/min, andat the second claw cut, to 7 μl/min could be achieved (cf. FIG. 8). Theaddition of antithrombin III should prevent a generation of thrombinfrom prothrombin due to the enzyme factor Xa in the administeredsolution.

EXAMPLE 16

Thrombogenicity of the preparation according to the invention

The individual components and mixtures thereof, of the preparationaccording to the invention were tested for their thrombogenic activityby using the method described by Wessler, J. Appl. Phys. 14:943-946(1959) in the venous blood subject to stasis in rabbits.

Rabbits were anesthesized with pentobarbital, the vena jugularis of theanimals was dissected and provided with loose ligatures at a distance of1-2 cm. The substances to be tested were injected into the animals, intothe ear vein opposite the dissected vena jugularis. The injection waseffected within 15 seconds. After a waiting time of 10-15 seconds, thevein segment was clamped. After further 10 minutes, the clamped veinsegment was removed and cut open in a citrated buffer in a Petri dish,and the thrombi obtained were evaluated between 0 and 4 by means of ascore (cf. table)

    ______________________________________                                        Degree of Thrombosis   Score                                                  ______________________________________                                        no formation of thrombi                                                                              0                                                      few small thrombi      0.5-1                                                  few middle-sized and many small thrombi                                                              2                                                      many middle-sized thrombi                                                                            3                                                      few large thrombi        3.5                                                  one coherent thrombus  4                                                      ______________________________________                                    

Each substance was tested on six animals each receiving 75 U of factorII/kg, 0.55 U of factor Xa/kg and 75 mU of antithrombin III/kg eitherseparately or in combination. The following table shows the mean valueof the Wessler score of six examined animals each. Pure citrated bufferwhich was also used as a dilution buffer was used as the control.

    ______________________________________                                        Test Substance                                                                Factor II                                                                              Factor Xa  Antithrombin III                                                                          Wessler Score                                 ______________________________________                                        +        -          -           0.17                                          -        +          -           0.17                                          -        -          +           0.08                                          +        +          -           0.25                                          +        -          +           0.08                                          -        +          +           0.17                                          +        +          +           0.17                                          -        -          -           0.2                                           (Bufer)  (Buffer)   (Buffer)                                                  ______________________________________                                    

It proved that none of the components used, neither separately nor incombination, had a thrombogenic activity in rabbit.

EXAMPLE 17

Action of the preparation according to the invention in dependence onthe form of administration

A preparation according to Example 6 which contained factor II andfactor Xa was each tested for its efficacy in 6 rabbits with inducedfactor VIII inhibitor hemophilia (cf. Example 14). The dose examined wasas described in Example 15. As the control, the animals were infusedwith pure buffer.

In the first test, an infusion of the test substances was carried outfor 30 minutes, corresponding to approximately 15 min/kg body weight, bymeans of an automatic infusion pump, at an infusion rate of 1 ml/min. Ina second test, the same dose, yet given as a bolus within 30 seconds ina small injection volume of 2.5 ml/kg body weight was administered tothe animals. As in Example 14, the bleeding intensity was measuredduring and after substance administration. It proved that both, at slowinfusion with a large infusion volume, and also at rapid injection of asmall volume dose, but with identical doses relative to the body weight,the pathologically extended bleeding intensity of 67 μl/min could benormalized to 5 μl/min, which corresponds to the bleeding behavior ofthe healthy control animals. By administration of buffer no change ofthe bleeding intensity was found with either of the two infusionmodalities. Despite the rapid injection as a bolus, no intolerancereaction could be observed during injection, and neither so during slowinfusion. For commercially available preparations of comparableindication (activated prothrombin complex preparations), infusion ratesin the range of <0.05 ml/min/kg body weight are recommended to avoidacute thromboembolic side effects and intolerance reactions. As the testindicated, due to its high purity the preparation of the invention couldbe administered without any side effects at 5 ml/min/kg body weight,i.e. at the 100-fold injection rate.

EXAMPLE 18

Action of partial prothrombinase in rats exhibiting a platelet defect

Fawn-Hooded rats described by T. B. Tschopp and M. B. Zucker (HereditaryDefect in Platelet Function in Rates, Blood 1972; 40:217-226) are usedas the thrombocytopathy model. This rat strain which had been furthercharacterized by S. L. Raymond and W. J. Dodds (Characterization of theFawn-Hooded Rats as a Model for Hemostatic Studies, Thrombos Diathes.haemorrh. 1975; 33:361-369) is characterized by an abnormally increasedbleeding time and a reduced platelet aggregation, while prothrombintime, partial thromboplastin time, plasma factor VIII and fibrinogenlevels as well as platelet number are in the same range as that of thehealthy rat. The thrombopathy occurring in the fawn-hooded rat isassociated with a reduced thrombin-induced ATP and ADP release, as wellas with a reduced serotonin release and thus is characterized as"storage pool deficiency" reflecting the "storage pool deficiency" inman.

The fawn-hooded rats are anesthesized with ketamine- xylazine (100mg/kg+5 mg/kg) i.m. A catheter is placed into a jugular vein, via whichthe substances to be tested are infused. Subsequently, the bleeding timeis determined, Simplate R disposable blood lancet (Organon, Technica)being used therefor. At a distance of approximately 1.5 cm from theradix of the tail, one dorsal, and subsequently one ventral longitudinalsection are made and the bleeding time is measured. The mean value ofthe two individual measurements is taken as the bleeding time. Then thetest substance (buffer, factor II according to Example 2, factor Xaaccording to Example 4, and partial prothrombinase according to Example6) is applied; after 30 minutes, the bleeding time is determined again.Healthy Sprague-Dawley rats (Charles River) are used as control.

Testing was effected in groups of 10 animals each, the complex of thepartial prothrombinase being administered in 2 doses, 75 U/kg bodyweight (BW) prothrombin and 0.55 U/kg BW factor Xa as well as 150 U/kgBW prothrombin and 1.1 U/kg BW factor Xa. As the control, the individualcomponents and buffer are administered. The mean bleeding times measuredin the individual test groups are given in the following table. Healthyrats have a bleeding time of 168±5 seconds under the same testconditions, while rates suffering from thrombopathy have an increasedbleeding time of 335±10 seconds. By administering partialprothrombinase, this abnormally increased bleeding time can be reducedto approximately 270 seconds.

                  TABLE                                                           ______________________________________                                        Bleeding time (sec)                                                           Mean value from                                                                            Factor II (U/kg)                                                 10 animals   0         75         150                                         ______________________________________                                        FXa     0        335 ± 10                                                                             296 ± 13.sup.ns                                                                     307 ± 11.sup.ns                        (U/kg)  0.55     n.d.      273 ± 13*                                                                           n.d.                                              1.1      320 ± 13.sup.ns                                                                      n.d.     268 ± 11***                            ______________________________________                                    

Statistical evluation:

The data are given as mean values ± standard deviation. The group meanvalues were compared by means of the Student T test.

EXAMPLE 19

Action of partial prothrombinase in von Willebrand factor-deficient dog

A dog exhibiting congenital von Willebrand factor deficiency havingnon-measurable von Willebrand factor activity and antigen plasma levelsand a factor VIII plasma concentration that is reduced by 50% is treatedwith a partial prothrombinase at a dose of 100 U/kg BW. For this, thedog is anesthesized and subsequently the partial prothrombinase,produced according to Example 6, is given intravenously as a bolus.Immediately before administration of the test substance as well as 15min, 30 min, 1 h, 2 h, 3 h, 24 h and 48 h after infusion, blood samplesare taken, plasma is produced therefrom, and the prothombin, thrombinand factor VIII concentrations are determined in the plasma samples.

Before administering the partial prothrombinase as well as 3 and 24 hafter injection, the cuticle bleeding characteristics are determined.For this, the method of A. R. Giles, S. Tinlin and R. Greenwood, ACanine Model of Hemophilic (Factor VIII:C Deficiency) Bleeding, Blood1982; 60, is used in a modified form. The fur surrounding the claw isshaved to prevent blood emerging at the later bleeding from beingabsorbed by the fur. The cuticle is injured by means of a nail clipper.Immediately thereafter, filters (Pipetman P5000 protection filter,Gilson) are established below the wound such that blood can dripdirectly onto the filter without being sucked up thereby on account of acapillary effect, so as to prevent a forming blood clot from beingdestroyed. The filter units are exchanged every 2 minutes, and theemerging blood is collected in fractions. Blood is continued to becollected for 30 minutes, and then the wound is cauterized if thebleeding has not stopped. Different claws of one and the same animal canbe used.

To quantitate the bleeding characteristic, the blood collected infractions on the filters is each extracted with 5 ml of a 0.04% ammoniumhydroxide solution for 5 h. Thereby the erythrocites, which have beencollected in the filter with the blood, are lysed. By a 10 minsonication (Sonorex RK 100, Bandelin electronic, Berlin), the hemoglobinis extracted and quantitated photometrically at 416 nm against acalibration curve. A calibration curve can be established by pipettingcanine blood in volumes of between 10 μl and 1 ml onto filters,extracting the filters as described above and photometricallydetermining the hemoglobin at 416 nm. Correspondingly, linearcalibration curves can be established which enable the hemoglobinconcentration to be calculated directly for the amount of blood perfilter. The bleeding characteristics of the cuticle cutting aredetermined by graphically plotting the amounts of blood per 2 minfraction against time. For an evaluation of the bleedingcharacteristics, the accumulated blood loss is determined by entering inthe graph the individual blood fractions additively against time. Theincrease of the accumulated bleeding between 10 and 20 min is taken as arelevant bleeding criterion. This value is independent of the initialblood amount which may be subject to variations because of poorlystandardizable claw cutting techniques. The increase of the bleedingcharacteristic in 10 to 20 min observation intervals serves as a measurefor the intensity of bleeding and is stated in ml blood/min. An increaseequal zero, corresponding to 0 ml/min, means that the bleeding hasstopped; an increase >0 with a correlation coefficient of >0.8 meansthat there is a constant bleeding. Normal dogs tested under theseconditions do not exhibit a bleeding in the observation interval, i.e.the bleeding has already previously come to a stop (bleeding intensity:0.0 ml/min).

Prior to administration of the partial prothrombinase, the bleedingintensity was 1.05 ml/min, and after 3 h it was reduced to 0.35 ml/min,and after 24 h it was further reduced to 0.47 ml/min.

The factor VIII plasma level remained constant over the entire period ofobservation, von Willebrand factor antigen remained below the detectionlimit, prothrombin was at 0.7 U/ml before substance administration androse to 2.4 U/ml after injection of the partial prothrombinase and waseliminated from circulation with a half-life of approximately 24 h. 1 hafter administration of the test substance, a significant thrombingeneration could be determined with a chromogenic substrate for thrombinTh1 (IMMUNO). Injection of partial prothrombinase was tolerated by thedog without any side effects.

EXAMPLE 20

Action of partial prothrombinase and of von Willebrand factor in the vonWillebrand factor-deficient dog

Analogous to Example 19, the dog was treated with a partialprothrombinase at a dosage of 100 U/kg BW and a purified von Willebrandfactor at a dosage of 60 RCOF U/kg BW. Administration of thiscombination lead to a complete normalization of the abnormally increasedbleeding behavior after 24 h in the von Willebrand factor-deficient dog,the plasma parameters being comparable as in Example 19 relative toprothrombin and thrombin. Therebeyond, there was an endogenous factorVIII increase to approximately 200% of the initial value, which remainedconstant over a period of more than 48 h and subsequently returned tothe initial value over a period of approximtely 120 h. Von Willebrandfactor was eliminated with a half-life of 20 h. From the literature (L.Drouet, J. Roussi, M. Bonneau, G. A. Pignaud, P. L. Turecek, F. Dorner,U. Schlokat, F. G. Falkner, B. Fischer, A. Mitterer and H. P. Schwarz,The effect of recombinant human von Willebrand Factor in pigs withsevere von Willebrand Disease. Blood 1995; 86: 612a-AbsNo.2435 (abs)) ithad been known that the administration of von Willebrand factor aloneleads to an only partial normalization of the bleeding behavior.

EXAMPLE 21

Action of partial prothrombinase as an antagonist of peptideanticoagulants

A partial prothrombinase in a composition of 57 U of factor II and 1.2 Uof factor Xa was dissolved in 20 mM Tris-HC1-buffer containing 150 mMNaCl, pH 7.4, and incubated for 15 min at room temperature for theformation of the complex. Subsequently, an aliquot of 50 μl was takenand incubated for 90 seconds at 37° C. with 50 μl of a Tris-imidazolebuffer, pH 8.4. Subsequently, it was admixed with 100 μl of a solutionof the chromogenic substrate methoxy-carbonyl-D-cyclohexylalanyl-glycyl-L-arginine-p-nitroanilide-hydroacetate so that theconcentration of the chromogenic substrate was 1 mmol/l. Subsequently,the kinetics of the cleavage of the chromogenic substrate wasphotometrically determined at 405 nm for 3 min at 37° C. The chromogenicsubstrate which is hydrolyzed by both thrombin and factor Xa, had aΔOD/min of 0.084 when incubated with partial prothrombinase. As acontrol, factor Xa of the same concentration, yet without prothrombinwas used in this test, and there appeared a conversion rate of thechromogenic substrate of 0.064 ΔOD/min. Recombinant hirudin(Rhein-Biotech) at a concentration of 0.0025 U/ml was added to thepartial prothrombinase, and the conversion rate of the chromogenicsusbtrate was also investigated. It showed that no further substrateconversion could be measured after the deduction of the substrateconversion of pure factor Xa (0.064 ΔOD/min), which was due to acomplete neutralisation of the hirudin.

Beside the effective and specific thrombin inhibitor hirudin, then alsothe selective factor Xa inhibitor, recombinant Tick-anticoagulantpeptide, a recombinant equivalent of serin protease inhibitor ofornithodoros moubata whose anticoagulatory in vivo-efficacy had beendescribed by G. P. Vlasuk, D. Ramjit, T. Fujita et al. in Comparison ofthe In Vivo Anticoagulant Properties of Standard Heparin and the HighlySelective Factor Xa Inhibitors Antistasin and Tick Anticoagulant Peptide(TAP) in a Rabbit Model of Venous Thrombosis. Thrombos Haemostas 1991;65: 25W-262, was added at a concentration of 50 Fg/ml. Already 30 minafter the addition of the recombinant tick-anticoagulant peptide, noenzyme activity could be measured any longer with the chromogenicsubstrate. The test shows that partial prothrombinase is an efficientantagonist of the peptide anticoagulants hirudin and tick-anticoagulantpeptide.

What is claimed is:
 1. A method of establishing a supranormalprothrombin concentration in vivo in the blood of a patient, wherein themethod comprisesadministering an effective dose of purifiedprothrombinase factors to said patient, wherein said purifiedprothrombinase factors lack thrombin activity.
 2. The method accordingto claim 1, wherein said administering further comprises administrationof an effective dose of purified prothrombin.
 3. The method according toclaim 1, wherein said administering further comprises administration ofan effective dose of purified prothrombin and purified factor Xa.
 4. Themethod according to claim 1, wherein a supranormal concentration of atleast 1.5 U of prothrombin/ml blood is established.
 5. The methodaccording to claim 1, wherein a supranormal concentration of at least 2U of prothrombin/ml blood is established.
 6. The method according toclaim 1, wherein the purified prothrombinase factors are administeredwith a double double-chamber syringe.
 7. The method according to claim3, wherein the purified prothrombin and factor Xa are administered witha double double-chamber syringe.
 8. The method according to claim 1,wherein the administering includes an effective dose of at least one offactor V or factor Va.
 9. The method according to claim 1, wherein theadministering does not include phospholipids.
 10. The method accordingto claim 1, wherein the administering does not include any free calciumions.
 11. The method according to claim 10, wherein the administeringfurther comprises administration of a calcium chelating agent.
 12. Themethod according to claim 1, wherein the administering further comprisesadministration of magnesium ions.
 13. The method according to claim 3,wherein the effective dose comprises factor Xaβ.